Thiamin is an essential cofactor in all living systems and is a required component of the human diet. Short-term deprivation of thiamin results in beri beri and Wernicke's encephalopathy and prolonged deprivation is lethal. Thiamin is also an important commercial chemical; it is widely used as a food additive and as a flavoring agent and annual production is on the order of 3,300 tons. Thiamin pyrophosphate, the active form of vitamin B1, plays an important role in carbohydrate metabolism and in branched-chain amino acid metabolism where it stabilizes acyl carbanion intermediates. Much of the biosynthetic pathway for prokaryotes is well understood. In B. subtilis, thiamin pyrophosphate is synthesized from glycine, deoxy-D-xylulose 5-phosphate, cysteine and aminoimidizaole ribotide. The biosynthetic pathway is complex and uses 14 gene products. We have now reconstituted the biosynthesis of the pyrimidine synthase and are just beginning to unravel the mechanistic details of this complex arrangement. In yeast, thiamin pyrophosphate is synthesized from glycine, cysteine, NAD, PLP and histidine and requires four enzymes. We now have basic understanding of the thiazole synthase, but little is known about the pyrimidine synthase. We have also discovered a new thiamin salvage pathway and are beginning to explore thiamin transport. Finally, the sulfur transfer system first discovered in the biosynthesis of the thiazole in prokaryotes is likely to be widespread. Our proposal has five specific aims. In aim 1 we will continue to study the formation of the thiazole moiety by determining the structures of ThiH, the ThiG/ThiS covalent adduct and complexes of TenI and Thi4 with bound ligands. In aim 2 we will study the formation of the pyrimidine by determining the structures of Thi5 and of iron-reconstituted ThiC with bound ligands. In aim 3, we will study the thiamin phosphate synthase by determining the structures of Thi6 and ThiE complexed with the recently identified carboxylthiazole. We will also investigate ThiE as a target for the design of antimicrobial agents. In aim 4 we will study thiamin salvage and uptake by determining structures of TenA orthologs, ThiY, YlmB, Thi20 (TenA/ThiD fusion) and newly identified thiaminase I orthologs. We will also determine the structures of enzymes involved in oxythiamin salvage. In aim 5, we will investigate the generalization of the thiamin sulfur transfer system. We will determine the structures of related proteins in thioquinilobactin biosynthesis and of proteins in a novel pathway from sulfate to thiocarboxylate. For all the systems described above, we will collaborate with Tadhg Begley to understand the biochemistry and mechanistic enzymology. These studies will result in (1) an understanding of the biosynthesis of a vitamin required for all forms of life, (2) a mechanistic understanding of the unprecedented chemistry used for thiamin biosynthesis, and (3) approaches for the construction of overexpression strains that can be used for the commercial production of thiamin by fermentation. PUBLIC HEALTH RELEVANCE: This research studies the biochemical pathways by which organisms synthesize, salvage and transport thiamin (vitamin B1). The research has implications for the commercial production of thiamin using fermentation, and for the development of antimicrobial agents that target thiamin biosynthesis.